The goal of this research project is to structurally characterize the origins of DNA replication in the bacterium EScherichia coli, the bacteriophage T4, and the eucaryotic slime mold Physarum polycephalum and to study the mechanisms by which replication is initiated at these origins. A 9 kb EcoRl restriction fragment that carries the origin of E. coli have been identified and has been cloned. This EcoRl fragment will now be combined with a fragment of plasmid R6K DNA which carries temperature sensitive replication functions to produce a hybrid plasmid capable of replicating either from the E. coli origin or the R6K origin depending upon growth conditions. This will allow E. coli origin mutants to be selected and maintained, thus providing a way to determine which nucleotide sequences at the E. coli origin are important for function. To insure a totally accurate determination of the normal nucleotide sequence at the E. coli origin of replication, origin DNA isolated directly from the E. coli chromosome will also be sequenced. The response of cell growth to the presence of hybrid plasmid carrying various portions of the origin region will be examined in order to better understand control of replication and cell division. A study of the origin region's apparent association with the cell membrane will be continued, using restriction endonucleases to remove DNA fragments not specifically bound to membrane in isolated DNA-membrane complexes. The work with T4 and Physarum involves the construction of physical maps and the dissection of partially replicated, radioactively puse-labeled chromosomes with restriction endonucleases in order to identify origin-containing fragments for further study. In Physarum the timing of replication within neighboring replicons will be examined for a selected segment of DNA so as to more clearly define the manner in which replication is controlled throughout the S period in lower eucaryotes.